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Published ahead of print on May 1, 2003, doi:10.1165/rcmb.2002-0044OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 29, pp. 513-522, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2002-0044OC

Interleukin-11 and Interleukin-6 Protect Cultured Human Endothelial Cells from H2O2-Induced Cell Death

Aaron B. Waxman, Keyvan Mahboubi, Roy G. Knickelbein, Lin L. Mantell, Nicholas Manzo, Jordan S. Pober and Jack A. Elias

Pulmonary Critical Care Unit, Massachusetts General Hospital, Boston, Massachusetts; Interdepartmental Program in Vascular Biology and Transplantation, Boyer Center for Molecular Medicine, Yale University School of Medicine; Department of Pediatrics, Yale University School of Medicine, Department of Internal Medicine, New Haven, Connecticut; Cardiac Research Laboratory, Department of Surgery, North Shore-Long Island Jewish Medical Center, New York University School of Medicine, Manhasset, New York; and Section of Pulmonary and Critical Care Medicine, Yale University School of Medicine, Department of Internal Medicine, New Haven, Connecticut

Address correspondence to: Aaron B. Waxman, Pulmonary Critical Care Unit, Massachusetts General Hospital, Harvard Medical School, 55 Fruit Street, Bulfinch 148 Boston, MA 02114. E-mail: ABWaxman{at}Partners.org


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Acute lung injury is a frequent and treatment-limiting consequence of therapy with 100% oxygen. Previous studies have determined that both interleukin (IL)-6 and IL-11 are protective in oxygen toxicity. This protection was associated with markedly diminished alveolar–capillary protein leak, endothelial and epithelial membrane injury, lipid peroxidation, and pulmonary neutrophil recruitment. Hyperoxia also caused cell death with DNA fragmentation in the lungs of transgene (-) animals, and both IL-6 and IL-11 markedly diminished this cell death response. However, the mechanism(s) by which these cytokines protect cells from death is unclear. In the present study, we characterized the effects of H2O2 on subconfluent human umbilical vein endothelial cell (HUVEC) and human pulmonary microvascular endothelial cell (HPMEC) cultures. We found that preincubation of HUVEC cultures with either IL-6 or IL-11 diminished H2O2 (1.0 mM)-induced cell death. Similar effects were noted with HPMEC showing that this effect is not HUVEC-specific. The protective effects of both IL-6 and IL-11 were not associated with any changes in antioxidants and were decreased by ~ 80% in the presence of U0126, a specific inhibitor of MEK-1–dependent pathways. The cytoprotective effects of IL-11 and IL-6 were also completely eliminated in STAT3 dominant-negative transduced HUVEC cultures. These studies demonstrate that IL-6 and IL-11 both confer cytoprotective effects that diminish oxidant-mediated endothelial cell injury. They also demonstrate that this protection is mediated, at least in part, by a STAT3 and MEK-1–dependent specific signal transduction pathway(s).

Abbreviations: human pulmonary microvascular endothelial cell(s), HPMEC • horseradish peroxidase, HRP • human umbilical vein endothelial cell(s), HUVEC • interleukin, IL • polyacrylamide gel electrophoresis, PAGE • phosphate-buffered saline, PBS • sodium dodecyl sulfate, SDS • superoxide dismutase, SOD


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Supplemental oxygen is commonly used to enhance tissue oxygen delivery and minimize the adverse effects of hypoxia. Supplemental oxygen is also a major stimulus for the generation of reactive oxygen species. This is a pressing issue when patients require 100% oxygen, which rapidly causes hyperoxic acute lung injury with endothelial and alveolar epithelial injury and increased pulmonary capillary permeability. Hyperoxic injury is caused, in great extent, by the excessive production/accumulation of oxygen-free radicals and reactive nitrogen species at subcellular sites (1). Oxidant-mediated lung injury is characterized by: (i) the loss of capillary endothelial cells with capillary leak, (ii) inflammation of the interstitium with thickening of the alveolar–capillary membrane, (iii) hyperplasia of type II pneumocytes, and (iv) injury and rounding of type I pneumocytes. The pulmonary capillary endothelium appears to be the earliest and a major site of injury with epithelial damage occurring at later time points (2, 3).

Interleukin (IL)-6 is a pleiotropic cytokine that is produced at sites of tissue inflammation. It is classified as an IL-6–type cytokine with IL-11, leukemia inhibitory factor, Cardiotrophin-1, Oncostatin M, and ciliary neurotrophic factor based on the overlapping effector profiles of these cytokines and their shared use of gp130 as the ß-subunit in their multimeric receptor complexes (4). Recent studies have demonstrated that IL-6 also has potent anti-inflammatory and protective properties (5, 6), and inhibits apoptosis (7). Using transgenic mice in which IL-6 was selectively overexpressed in the lung (CC10–IL-6 mice), we recently demonstrated that IL-6 has impressive protective effects in the setting of 100% O2–induced acute lung injury. This tolerance is manifested as enhanced survival, decreased pulmonary edema and alveolar–capillary protein leak, and decreased lung lipid peroxidation when compared with transgene (-) controls. Furthermore, transgene (-) mice manifest an impressive cell death response associated with DNA fragmentation, which was inhibited in the IL-6 transgene (+) animals (8).

IL-11 is a 20-kD cationic member of the IL-6–type cytokine family, which has been demonstrated to confer tissue cytoprotection in a variety of tissues including the bowel (9–11) and lung (12–14). In vascular endothelial cells, IL-11 pretreatment resulted in resistance to immune-mediated injury without inhibiting proinflammatory responses (15). Previous studies from our laboratory demonstrated that the transgenic overexpression of IL-11 in the lung caused a remarkable tolerance to the toxic effects of 100% oxygen, with transgene (+) animals demonstrating remarkably enhanced survival, and decreased lung lipid peroxidation, neutrophil recruitment, alveolar–capillary protein leak, pulmonary edema and hyperoxia-induced cell death, and DNA fragmentation (16).

In the present study, we characterized the effects of IL-6 and IL-11 on the H2O2-mediated injury of human umbilical vein endothelial cell (HUVEC) and human pulmonary microvascular endothelial cell (HPMEC) cultures. We found that preincubation of vascular endothelial cell cultures with either IL-6 or IL-11 diminished H2O2-induced cell death. We also noted that these protective effects of IL-6 and IL-11 were not associated with changes in antioxidants that explain these responses, and could be abrogated in the presence of MAPK inhibitors or in STAT3 dominant-negative endothelial cultures. These studies demonstrate that both IL-6 and IL-11 have cytoprotective properties that diminish oxidant-mediated endothelial cell injury. They also demonstrate that this protection is mediated by MEK- and STAT3-dependent signal transduction pathways.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Antibodies, Drugs, and Cytokines
Rabbit polyclonal Abs reactive to STAT3 and phosphotyrosine-STAT3, p42 and p44, p38, and pSAPK were purchased from New England Biolabs Inc. (Beverly, MA). All reagents used in the antioxidant assays were purchased from Sigma (St Louis, MO). The pharmacologic inhibitor of mitogen-activated protein/extracellular signal–related kinase kinase (MEK-1) U0126 were obtained from Calbiochem (La Jolla, CA). Recombinant human IL-6 and monoclonal anti-human IL-6R Ab were purchased from R&D Systems Inc. (Minneapolis, MN). Recombinant human IL-11 was provided by Genetics Institute (Andover, MA).

Cell Culture
Cells and reagents. HUVEC and HPMEC were obtained from Clonetics (San Diego, CA) and cultivated in endothelial growth medium supplemented with 2% fetal bovine serum, bovine brain extract (3 mg/ml), hEGF (10 mg/ml), hydrocortisone (1 mg/ml), Gentamicin (50 mg/ml), and Amphotericin-B (50 µg/ml). Cells were used between passages 2 and 5. For the evaluation of IL-6R, both endothelial cells obtained from Clonetics as described above, and Human EC directly isolated from umbilical veins in our laboratory as previously described (17, 18) were used. Human EC were cultured on gelatin (J. T. Baker, Phillipsburg, NJ)-coated tissue culture plastic at 37°C in 5% CO2–humidified air in Medium 199 containing 20% fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin (all from Life Technologies, Grand Island, NY), 50 µg/ml of FGF-1 (Collaborative Research/Becton Dickinson, Bedford, MA), and 100 µg/ml of porcine intestinal heparin (Sigma). Cultured cells were treated with either IL-6 or IL-11 once the cultures were confluent. To assure confluence, cultures were used 72 h after they were judged to be confluent. Cells were incubated with the cytokines for 24 h at 37°C, after which the medium was removed and replaced with standard growth medium with the noted concentration of H2O2. After an additional 1-h incubation period the cells were evaluated as described below.

Cell Viability Assays
Trypan blue exclusion. Cells in culture suspension were exposed to Trypan blue dye (0.04% in phosphate-buffered saline [PBS]) after exposure to H2O2, placed on a hemocytometer, and examined using a light microscope. Two hundred random cells were counted after each treatment, and the percentage of blue (dead) cells was expressed as the percentage of viable cells for any given condition. As an additional test of viability after exposure to H2O2, media was removed and the cells rinsed with fresh incubation media. The cultures were returned to the incubator with fresh media and allowed to grow again to confluence.

DAPI staining. As previously described (19), endothelial cells were harvested, spun onto slides, fixed with methanol, washed and incubated with 0.1 µg/ml, 4',6-diamidino-2-phenylindole, dihydrochloride (DAPI) (Molecular Probes Inc.) for 5 min. After incubation the slides are washed, dried, embedded in mounting medium, and examined with a fluorescence microscope.

Propidium iodide and Annexin V staining. Both floating and adherent cells were harvested 24–48 h after treatment; washed once with 1 ml of PBS, 5 mM EDTA; and fixed with 1 ml of 70% ethyl alcohol while vortexing gently. Fixed cells were stored at 4°C for 1 h to several days. Cells were pelleted by centrifugation, washed once with 1.0 ml of PBS, 5 mM EDTA, and resuspended with 0.3–1.0 ml Propidium Iodide (PI) mix (250 µg/ml PI, 5 µg/ml RNase A, 1x PBS, and 5 mM EDTA). PI is impermeable to live cells and early apoptotic cells, but enters necrotic cells and stains their nuclei with red fluorescence. After incubation in the dark for 1 h at room temperature, the cells were analyzed on a Becton Dickinson FACscan, and apoptotic (sub-G1 population) and necrotic cells were quantified.

Immunoblotting. Cells were washed twice with ice-cold PBS containing 1 mM sodium orthovanadate and 1 mM sodium fluoride, and lysed with ice-cold RIPA lysis buffer (PBS, 1% NP40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS], 1 mM PMSF, 10 µg/ml leupeptin, 1 mM sodium orthovanadate). Cell lysates were clarified by centrifugation at 10,000 x g for 15 min, and protein concentrations of supernatant were determined by using a Bio-Rad assay kit (Bio-Rad, Hercules, CA). Lysates were prepared for SDS-polyacrylamide gel electrophoresis (PAGE) by adding an equal volume of 2x SDS-PAGE sample buffer (100 mM Tris-Cl, pH 6.8, 200 mM dithiothreitol, 4% SDS, 0.2% bromphenol blue, 20% glycerol) and heating the mixture in a boiling water bath for 3 min. Twenty micrograms of protein was separated on SDS-PAGE and transferred onto a polyvinylidene difluoride membrane by electrophoresis (Immobilon P; Millipore, Bedford, MA). After blocking with tris-buffered saline tween (10 mM Tris-HCl, pH 8.0, 0.150 mM NaCl, 0.05% Tween 20) containing 5% milk for 1 h at room temperature, the membranes were incubated with blocking solution containing the indicated Ab overnight at 4°C. Membranes were washed and incubated with a suitable horseradish peroxidase (HRP)-conjugated detecting reagent (Jackson Immuno Research, West Grove, PA), and HRP activity was detected using an enhanced chemiluminescence kit according to the manufacturer's instructions (Pierce, Rockford, IL). Exposed films were scanned using a laser densitometer (Fast Scan, Series 300; Molecular Dynamics, Sunnyvale, CA).

Antioxidant assays. Catalase activity was determined using the method of Aebi (20). Immediately after preparation, 15–30 µg of protein was added to 10 mM H2O2, and the decrease in absorbance measured for 2 min at 240 nm. The rate of change in absorbance was converted to units of enzyme activity, determined from a standard curve generated each day using catalase purchased from Sigma. Enzyme activity was then standardized to mg protein.

Glutathione reductase activity was measured using the assay of Bellomo and coworkers (21). This assay is based on the reduction of glutathione disulfide (oxidized glutathione) (GSSG) to reduced glutathione (GSH) by glutathione reductase, using NADPH as a donor for H+. NADPH is absorptive at 340 nm. The assay follows the decrease in OD at this wavelength with time as NADPH is oxidized to nicotinamide adenine dinucleotide phosphate (NADP). The assay was performed using 50- and 100-ml aliquots of cell supernatant protein immediately after preparation (~ 100–200 mg protein) added to phosphate buffer containing excess GSSG and NADPH. The rate of change was determined at 340 nm for 5 min and converted to mUnits activity using a standard curve performed each day using glutathione reductase purchased from Sigma. Enzyme activity was standardized to mg protein.

Glutathione peroxidase activity was determined using the assay described by Flohe and Günzler (22). In this assay, GSSG formed during glutathione peroxidase reaction is then continuously reduced by an excess of glutathione reductase activity, thereby maintaining a constant level of GSH. Glutathione reductase activity is dependent on NADPH, which is then oxidized to NADP. The decrease in NADPH, which is monitored at 365 nm, is dependent of the GSSG level, which, in turn, depends on the glutathione peroxidase activity. The assay was performed by adding 10–50 mg protein to phosphate buffer containing 10 mM GSH, 1.5 mM NADPH, and 0.24 U glutathione reductase. The reaction was initiated by adding H2O2 (150 mM final concentration), which acts as the H+ acceptor. The change in absorbance at 365 nm was measured for 5 min and converted to mUnits activity using a standard curve performed each day using glutathione peroxidase purchased from Sigma. Enzyme activity was normalized to mg protein.

Superoxide dismutase (SOD) enzymatic activity was determined using the assay described by McCord and Fridovich (23) and by Crapo and colleagues (24). In brief, the reduction of cytochrome C by xanthine/xanthine oxidase–generated superoxide anion (O2-) was monitored spectrophotometrically at 550 nm. SOD inhibits cytochrome C reduction by converting O2- to H2O2. The amount of endothelial cell protein lysate necessary for 50% inhibition was determined and defined as one unit of enzyme activity. This total SOD activity was a combination of MnSOD and CuZnSOD. To differentiate between these two enzymes, the assay was performed at both pH 7.8 and pH 10.2 to amplify the pH-sensitive CuZnSOD activity. CuZnSOD activity was then selectively inhibited with 1-mM potassium cyanide.

Statistical Analysis
In all experiments n is equal to 6. Where appropriate, data are expressed as means ± SEM. Data sets were examined by one- and two-way ANOVA, and individual group means were then compared with Student's unpaired t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Dose Response of Endothelial Cells Exposed to H2O2
Experiments were first conducted to define the effects of H2O2 exposure on endothelial cell cultures. Both HPMEC and HUVEC were grown to confluence in medium containing 2% serum, and were then exposed to varying concentrations of H2O2 (0.1–5.0 mM) for 1 h. Cell survival, based on Trypan blue exclusion, ranged from 88–27% after this treatment. At 1 mM H2O2, survival was ~ 45% in HUVEC (Figure 1). Quantitatively similar results were obtained using HPMEC (data not shown). Thus, this concentration of H2O2 was used for all subsequent experiments to provide a maximum dynamic range for quantifying protective versus harmful responses.



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  		Figure 1. Dose–response curve showing survival of HUVEC after exposure to varying concentrations of H2O2 ranging from 0.1 mM to 5.0 mM for 1 h. Cell survival based on trypan blue exclusion ranged from 88–27%.

 
Effect of IL-6 and IL-11 on H2O2-Mediated Endothelial Cell Death
Initial experiments were undertaken to assess the optimal time period for pretreatment of endothelial cell cultures with each cytokine. Viability was assessed after 1, 4, 8, and 24 h. There was no significant difference in survival at each time point (data not shown). Subsequent viability experiments were done using 24-h preincubation. This time point was chosen because it has been a standard approach in our laboratory for assessing response to cytokine stimulation and to maximize any differences in effect. To determine if IL-6–type cytokine conferred protection in the setting of H2O2 exposure, HPMEC were incubated with varying concentrations of either IL-6 or IL-11 for 24 h and then exposed to 1 mM H2O2 for 1 h. IL-6 significantly improved cell survival at all cytokine concentrations that were assessed (5–200 ng). In contrast to the 45% survival of control medium–exposed cells, the survival of IL-6–treated cells ranged from 76–87% (Figure 2A). IL-11 (5–200 ng) conferred a similarly impressive survival benefit with 68–85% of HPMEC surviving H2O2 (1 mM) exposure (Figure 2B). Similar effects were seen with HUVEC. In general, the protective effects of IL-6 and IL-11 were more pronounced on HPMEC.



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  		Figure 2. (A) Survival of HPMEC after pretreatment with different doses of IL-6. (B) Survival of HPMEC after pretreatment with different doses of IL-11. In all cases, the asterisk indicates significantly enhanced survival compared with controls (P < 0.005).

 
Differentiation of Apoptosis and Necrosis
To further assess the cytoprotective effects of both IL-6 and IL-11, studies were undertaken to define the types of cell death induced by H2O2 in HUVEC cells. In our initial experiments this was done by assessing Annexin V and PI incorporation by flow cytometry. This allowed us to gate on cells with just an increase in Annexin V staining (early apoptosis), just an increase in PI staining (necrosis), or an increase in both Annexin V and PI (combined necrosis or late apoptosis). Both apoptosis and necrosis were induced by H2O2 in these experiments (Figure 3A). More cells also incorporated PI at later times. In addition, conditions that increased the percentage of cells exhibiting necrosis decreased the percentage of cells undergoing apoptosis (data not shown). In control cells (Table 1), 90% (± 1.0) failed to bind Annexin V or show PI uptake, 8% (± 1.0) had an isolated uptake of Annexin V, 0.3% (± 0.09) had an isolated uptake of PI, and 1.46% (± 0.09) of cells bound Annexin and took up PI. In contrast, after 1 h of exposure of HUVEC to 1 mM H2O2, 30% (± 4.7) of cells failed to stain with Annexin V or PI; 58% (± 3.5) of cells bound Annexin V only (consistent with pure apoptosis); 0.8% (± 0.03) of cells were only positive for PI (consistent with pure necrosis); and 11% (± 0.09) of cells bound both Annexin V and PI (consistent with late apoptosis and/or necrosis). The extent of necrosis was dose-dependent, with higher percentages of cells incorporating PI after exposure to high concentrations of H2O2. When HUVEC were pretreated with 100 ng/ml of IL-11 and then exposed to 1 mM H2O2 75% (± 8.6) of cells did not bind either Annexin V or PI; 19.0% (± 4.5) of cells bound Annexin V only; 0.4% (± 0.07) of cells bound only PI; and 5.7% (± 1.4) of cells bound both Annexin V and PI. Similarly, when HUVEC were pretreated with 100 ng/ml of IL-6 and then exposed to 1 mM H2O2 64% (± 1.2) of cells had no uptake of either Annexin V or PI; 30% (± 1.1) of cells had an isolated increase in Annexin V; 0.7% (± 0.03) of cells had an isolated uptake of PI; and 4.7% (± 0.25) of cells bound both Annexin V and PI. Similar findings were noted with HPMEC (data not shown). These data show that there is a significant decrease in the number of cells with features of apoptosis following exposure to H2O2 and pretreatment with either IL-6 or IL-11 compared with control (P < 0.05). This demonstrates that apoptosis is a major mode of cell death in cells exposed to 1 mM H2O2, and that both IL-11 and IL-6 protect cells from this cell death response. Additional support for the importance of apoptosis in this response was seen in assays of caspase-3 and assessment of DAPI images. Caspase-3 activity was induced 3.8-fold (over medium control) by exposure to H2O2 in these cells (Figure 3B). DAPI-fluorescence images were also compatible with an apoptotic response (Figure 3C).





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  		Figure 3. Exposure to H2O2 induces apoptosis. (A) Annexin V incorporation and PI incorporation was measured by flow cytometry to assess the mode of cell death. Relative fluorescence units represent the intensity of Annexin V incorporation (x-axis) and PI incorporation (y-axis). A shift to the right indicates phosphatidylserine translocation from the cytosolic surface to the extracellular surface, an indicator for cells undergoing apoptosis. An upward shift indicates PI incorporation, an indicator for cells undergoing necrosis and loss of membrane integrity. Cells treated with NP-40 were used as controls for necrosis. The figure is one representative experiment out of three experiments. (B) Comparison of caspase-3 activity between control HUVEC in room air, HUVEC treated with 1 mM H2O2, HUVEC pretreated with 100 ng/ml IL-6 and later exposed to H2O2 for 1 h, and HUVEC pretreated with 100 ng/ml IL-11 and later exposed to 1 mM H2O2. (C) DAPI-stained HUVEC demonstrating increased DAPI uptake with increased H2O2 concentration.

 

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  		TABLE 1 Differentiation of Apoptosis and Necrosis

 
Role of Antioxidants
Aerobic biologic systems have evolved several defense mechanisms to protect themselves from oxidative stress, including the redox cycling of glutathione (25) and catalase (26). To address the possibility that alterations in these defensive pathways might be responsible for the protection induced by IL-6 or IL-11, we compared these pathways in endothelial cell cultures treated with either IL-6 or IL-11. At baseline the activity of catalase, glutathione peroxidase, and glutathione reductase were similar between HPMEC and HUVEC cells. Exposure to H2O2 resulted in no significant change in either catalase or glutathione reductase. One-hour exposure to 1 mM H2O2 caused a 61% (442.4 ± 30.13 versus 169.3 ± 59) and 73% (315.4 ± 84 versus 83.6 ± 11) decrease in glutathione peroxidase activity in HPMEC and HUVEC, respectively. The addition of either IL-11 or IL-6 did not alter the activities of catalase, glutathione reductase, or glutathione peroxidase before or after H2O2 exposure (data not shown).

At baseline the activity of total SOD, Mn-SOD, and CuZnSOD was modestly increased in HPMEC compared with HUVEC (26.5 ± 0.34, 5.9 ± 1, and 20.6 ± 0.8 versus 18.7 ± 2, 3.5 ± 0.4, and 16.2 ± 2, respectively). Both IL-6 and IL-11 increased total SOD activity in both HPMEC and HUVEC. The increase was due to elevated activity of MnSOD, with little increase in CuZn SOD. In HPMEC cells, incubation with IL-6 for 24 h resulted in a 40% increase in MnSOD (6.5 ± 1 versus 8.1 ± 3), whereas stimulation for either 4 or 24 h with IL-11 resulted in a 33% and 27% (6.5 ± 1.0 versus 9.6 ± 1 and 8.9 ± 0.8, respectively) increase in MnSOD activity, respectively. None of the observed increases were statistically significant. Exposure to H2O2 alone for 1 h did not greatly affect SOD activity (6.5 ± 1 versus 6.1 ± 1). These data suggest that changes in antioxidants are not of a magnitude to account for the observed protection induced by either IL-11 or IL-6.

Role of STAT3 in IL-6 and IL-11 Protection
IL-6–type cytokine signaling frequently involves the phosphorylation of STAT3 and to a lesser extent STAT1 (15, 27). IL-11 induces tyrosine phosphorylation of STAT3 in HUVEC (15). To further define the signal transduction pathways that are involved in IL-6– and IL-11–induced protection of H2O2-treated HUVEC and HPMEC, we compared the protective effects of both IL-6 and IL-11 in STAT3 dominant-negative HUVEC cultures. STAT3 dominant-negative cultures were prepared as previously described (28). IL-6–induced protection was significantly decreased in STAT-3–deficient cells (Figure 4A). The protection seen with IL-11 pretreatment was also entirely blocked when these cells were treated with H2O2 (Figure 4B). We previously demonstrated that IL-11 phosphorylates STAT3 in HUVEC (15). To see if similar pathways were operative in HPMEC, we used Western blotting to determine whether IL-11 and IL-6 induces phosphorylation of STAT3 in pulmonary microvascular cells (Figure 5). No tyrosine phosphorylation was detected in unstimulated HPMEC by Western blot analysis. After 10 min of treatment of the cells with IL-11, there was a dose-dependent increase in phosphorylation of STAT3 (Figure 5). Western blotting of the same samples with anti-STAT3 antibodies demonstrated that the total levels of STAT3 were not changed by IL-11.



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  		Figure 4. Comparison of the survival of STAT3 dominant-negative HUVEC. In each case, survival was determined by Trypan blue exclusion. (A) Survival of STAT3 dn HUVEC exposed to H2O2 after pretreatment with IL-6. Striped bars, STAT3 dominant-negative HUVEC + IL-6; solid bars, HUVEC + IL-6. (B) Survival of STAT3 dn HUVEC exposed to H2O2 after pretreatment with IL-11. Striped bars, STAT3 dominant-negative HUVEC + IL-11; solid bars, HUVEC + IL-11. In each case, the asterisk indicates significantly enhanced survival compared with controls (P < 0.001). There was no significant difference in survival between STAT3 dominant-negative HUVEC treated with either IL-6 or IL-11 and controls.

 


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  		Figure 5. Dose-dependent activation of STAT3 by both IL-11 and IL-6 in HUVEC. The graph depicts the ratio of P-STAT3 to total STAT3 at each dose of cytokine for both IL-11 (closed circles) and IL-6 (open squares).

 
Our findings with IL-6 were initially surprising because previous studies reported that the IL-6R{alpha} is not present on HUVEC (29, 30). In contrast, we noted brisk IL-6–induced responses in these cells. To address this discrepancy, lysates from HUVEC and HPMEC were resolved on SDS-PAGE and immunoblotted with specific antibodies to IL-6R{alpha} as described in MATERIALS AND METHODS. To control for differences in culture conditions and potential mitogens, comparison was made between cells obtained from Clonetics and Human EC isolated from umbilical cords. To control for differences in receptor expression related to cell density, comparison was also made between cells grown to 85–90% confluence and cells grown to confluence. Immunoblots clearly demonstrated the expression of IL-6R{alpha}–chain protein in HUVEC (Figure 6). These receptors were functional because IL-6 phosphorylated STAT3 in HUVEC in a dose- and time-dependent fashion, and this phosphorylation was blocked by anti-human IL-6R{alpha} antibody (Figure 7).



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  		Figure 6. HUVEC express IL-6R-chain protein. Lysates from HUVEC were resolved on SDS-PAGE and immunoblotted with specific antibody to IL-6R (3 µg/ml) as described in MATERIALS AND METHODS. MW, molecular weight.

 


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  		Figure 7. Anti-human IL-6R antibody blocks STAT3 phosphorylation by IL-6 in HUVEC. HUVEC were incubated with either media alone (No Ab) or media containing anti IL-6R Ab (1 µg/ml) for 90 min. After incubation, cells were either untreated (control), treated with IL-6 (50 ng/ml), or IL-11 (50 ng/ml) for 15 min Lysates were resolved on SDS-PAGE and immunoblotted with specific Ab to either P-STAT3 or STAT3. Results were quantified by densitometry and are displayed in the graph. In each case, the bar represents the ratio of P-STAT3 to STAT3.

 
Role of MAPK and Effects of MAPK Inhibitor U0126 on Cytokine Mediated Protection
We previously demonstrated that stimulation of HUVEC with IL-11 results in a dose-dependent phosphorylation of p42/44 (15). To further understand the pathways that are involved in protection induced by IL-6 and IL-11, we compared the activation of the ERK, JNK, and p38 MAPK pathways in HPMEC. Cells were grown to confluence and studied using media with 2% FBS without additional growth factors. IL-6 and IL-11 did not increase the levels of phosphorylated p38 or pSAPK (data not shown). In contrast, a significant increase in the levels of P-p44/42 was readily appreciated. This response was maximal with a 15-min exposure to IL-6 or IL-11, and was blocked by U0126, a potent and specific inhibitor of MEK1 and MEK2 that blocks the production of inflammatory cytokines and matrix metalloproteinases (31) (Figure 8). U0126 completely abrogated the phosphorylation of p44/42 in HUVEC (Figure 8B) and HPMEC (data not shown) controls and HUVEC (Figure 8B) and HPMEC (data not shown) treated with H2O2.




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  		Figure 8. Both IL-11 (A) and IL-6 (B) significantly increased levels of P-p44/42 in a dose-dependent fashion. Neither IL-6 nor IL-11 stimulation resulted in increased levels of phosphorylated p38 or pSAPK (data not shown). This response was maximal with a 15-min exposure to IL-6 or IL-11, and was inhibited by U0126, an inhibitor of the ERK pathway. Results were quantified by densitometry and are displayed in the graphs. In each case, the bar represents the ratio of P-p44.42 to total p44.42. (C) The efficacy of U0126 was tested in both control HUVEC and HUVEC treated with H2O2. In both cases, U0126 completely blocked the phosphorylation of p44/42.

 
We then evaluated the protective effects of both IL-6 and IL-11 on both HUVEC and HPMEC incubated in the presence and absence of U0126, a specific inhibitor of MEK-1–dependent pathways. In these experiments, cells were grown to confluence and studied using media with 2% FBS without additional growth factors. U0126 decreased IL-6–induced protection by 10% (77.0 ± 1 versus 69.5 ± 0.7, P < 0.001) in HPMEC (Figure 9B). In contrast, U0126 decreased IL-11–induced protection by ~ 26% (70.7 ± 4 versus 52.5 ± 0.7, P < 0.001) in HPMEC (Figure 9B). Similar results were obtained in cultures of HUVEC incubated in the presence and absence of U0126 (data not shown). This suggests that both IL-11– and IL-6–induced protection of endothelial cells in vitro is mediated, at least in part, via a MEK-1–dependent signal transduction pathway. This pathway, however, appears to be more important in mediating the protective effects of IL-11.



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  		Figure 9. In each case, the column represents the percent survival of HPMEC exposed to H2O2 with or without cytokine pretreatment. Control cells were exposed only to H2O2. The column labeled U0126 shows the survival of HPMEC cells pretreated with U0126 and then exposed to H2O2. (A) The MEK1 inhibitor U0126 diminishes IL-6 protection of HPMEC. Striped bars, pretreated with IL-6; solid bars, pretreated with IL-6 + U0126. (B) The MEK1 inhibitor U0126 diminishes IL-11 protection of HPMEC. Striped bars, pretreated with IL-11; solid bars, pretreated with IL-11 + U0126. *P < 0.001 in comparison of cells treated with U0126 and both control(s).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The IL-6 family of cytokines reduces oxidant-mediated injury in vivo (8, 16). It is unclear whether these effects are predominantly anti-inflammatory, cytoprotective, or some combination of both. To test the hypothesis that cytoprotection plays an important role in IL-11– and IL-6–induced protection from oxidant-mediated cellular injury, we needed a biologically relevant system in which its cytoprotective effects could be evaluated independently of inflammation and other host responses. To establish such a system we characterized the effects of H2O2 on HUVEC and HPMEC cultures grown to greater than confluence. Using H2O2 (1.0 mM) to cause cell death, we found that preincubation of HUVEC cultures with either IL-6 or IL-11 diminished these cell death responses. Similar effects were noted with HPMEC, showing that this effect is not HUVEC-specific. In both cases there was no observed dose–response effect. The lack of dose response may be related to the 24-h preincubation period. It is possible that with a shorter exposure time there may be a more apparent dose–response effect. The protective effects of both IL-6 and IL-11 were not associated with changes in standard antioxidants that could explain this finding. However, they were abrogated by U0126, a specific inhibitor of MEK-1–dependent pathways and dominant-negative STAT3 constructs. This suggests that IL-6–type cytokine–induced protection of vascular EC is mediated, at least in part, via a STAT3 phosphorylation and an MEK-1–dependent signal transduction pathway.

The studies described are an extension of our previous reports on the protective effects of IL-11 (15, 16) and IL-6 (8). When IL-11 and IL-6 are expressed as a transgene in the airways of mice, they exhibit significantly improved survival during prolonged exposure to 100% oxygen. In both transgenic animal models the survival benefit was associated with diminished alveolar protein leak, lipid peroxidation, cellular membrane injury, and DNA fragmentation. IL-6 type cytokines have demonstrated protective effects in a variety of tissues. In the GI tract IL-11 decreases the injury seen with combined chemotherapy and radiation therapy (9, 11), hapten-induced colitis (32), ischemic colitis (10), and the inflammatory bowel disease of HLA-B27 transgenic mice (11). A number of studies have demonstrated that IL-11 can also protect at extra-abdominal sites because it enhances the survival of mice exposed to thoracic irradiation (12), decreases post–marrow transplant graft-versus-host disease (33), and ameliorates immune complex and endotoxin-induced pulmonary dysfunction (13, 14). IL-6 also has significant anti-inflammatory effects, including the ability to induce IL-1Ra synthesis, induce the release of soluble tumor necrosis factor receptors (5), and inhibit the production of the proinflammatory cytokines tumor necrosis factor and MIP2 (6, 34).

In addition to their anti-inflammatory properties, IL-6–type cytokines also have direct cytoprotective effects. IL-11 has been shown to protect clonogenic stem cells from radiation-induced injury (35), to inhibit apoptosis (36), and to accelerate the recovery of small intestinal mucosa in mice treated with combined chemotherapy and radiation (9, 36). IL-11 can also inhibit p53 protein expression in a murine model of bowel ischemia (10) and induce intestinal epithelial cell growth arrest (37). In vascular endothelial cells, IL-11 pretreatment resulted in resistance to immune-mediated injury without inhibiting proinflammatory responses (15). On the other hand, IL-6 can reduce cell death in a variety of systems and via different mechanisms, including the inhibition of superoxide production by chondrocytes (38) and the upregulation of Bcl2 family proteins (39, 40). When considered in combination, these data provide evidence that IL-6–type cytokine–mediated protective effects can be the result of direct cytoprotection as well as of the inhibition of tissue inflammation.

IL-11 and IL-6 use different ligand-binding proteins, but share a common signal transducer, gp130 (41). Cultured HUVEC express IL-11R{alpha} and gp130, and stimulation of HUVEC with IL-11 induces rapid phosphorylation of gp130, STAT3, and p42/p44 MAPKs (15). In contrast, IL-6R transcripts were previously reported to be undetectable by Northern blot analysis on human endothelial cells (29, 30), and IL-6 did not have a measurable effect on several endothelial cell functions (42, 43). We found that HUVEC and HPMEC have exuberant biologic responses to IL-6 and express IL-6R{alpha} protein. Furthermore, IL-6 induces rapid phosphorylation of STAT3 and p42/44 MAPKs in these cells. We also noted that these effects were abrogated by pretreatment with specific antibodies to the IL-6R. When viewed in combination, these studies demonstrate that HUVEC and HPMEC express IL-6R{alpha} and have fully functional IL-6 receptor complexes. This finding is in accord with what has been described for other IL-6–type cytokines, including IL-11 (15), and Oncostatin M (44).

The exact mechanism of how these cytokines are protective is still uncertain. The pretreatment of endothelial cells with either IL-6 or IL-11 results in phosphorylation of both p42/44 MAPK and STAT3. The JAK-STAT pathway is initiated by the binding of cytokines and growth factors to their specific receptors. Both the IL-6 and IL-11 receptor complexes lack intrinsic tyrosine kinase activity. Receptors for these cytokines associate with gp130. Gp130 associates with and activates JAKs, which in turn phosphorylate STAT proteins in the cytoplasm (45). Some STAT proteins such as STAT3 are phosphorylated at serine residues, possibly through MAPK pathways (46). It is likely that the activation of STAT3 either in concert with or coincident with activation of MAPK results in the production of cytoprotective proteins. Both IL-6 and leukemia inhibitory factor have been shown to mediate growth arrest and prevent apoptosis via STAT3 activation (47). Dominant-negative forms of STAT3 inhibited both IL-6–induced growth arrest and macrophage differentiation. IL-6 enhanced the growth of cells primarily through shortening the length of the G1 period, when STAT3 was suppressed (48). In these models, IL-6 generates both growth-enhancing signals and growth arrest– and differentiation-inducing signals at the same time. STAT3 may be a key molecule that determines the cellular decision from cell growth to differentiation or death. In fact, there is a strong correlation between elevated levels of antiapoptotic members of the Bcl-2 family and STAT3 activation (49). Human myeloma cell line M1 is dependent on IL-6 for survival, and constitutive STAT3 activation contributes to Bcl-x gene expression, whereas functional disruption of IL-6–mediated STAT3 signaling inhibits Bcl-x expression and correlates with induction of apoptosis (50). In a mouse model, IL-6–induced STAT3 activation was required for antiapoptotic signaling for T cell proliferation and prevention of apoptosis (51).

In summary, these studies demonstrate that IL-11 and IL-6 provide direct cytoprotection in an isolated in vitro endothelial cell system. They also show, for the first time, that functional IL-6 receptors are present on endothelial cells in culture, and that cytokine treatment of these cells mediates these protective effects via a STAT3- and, to a lesser extent, p42/p44 MAPK-dependent pathway. These studies provide insights into the mechanisms that underlie these important responses, and suggest that IL-6 and or IL-11 might be a useful therapeutic in diseases characterized by H2O2-induced endothelial cell death.


   Acknowledgments
 
The authors thank Pasquale Razzano, M.S., and William Franek, M.S., for their excellent technical assistance. This work was supported by grants RO1-HL-64242 (J.A.E.), K08-HL-03888 (A.B.W.), and RO1-HL-62188 (J.S.P.).

Received in original form April 19, 2002

Received in final form April 23, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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